Biopharmaceutical drugs, especially therapeutic antibodies, are produced by mammalian cell culture. Chinese hamster ovary (CHO) cells are the most commonly used host cell. These production systems are prone to adventitious and endogenous virus infection, which presents a potential safety problem for the biopharmaceutical drug. Viral clearance procedures and viral load measurements are therefore used to promote the safety of the drug. Steps employed to reduce viral load include nanofiltration, virus inactivation by heat or pH hold, and chromatography. Viral load and the effectiveness of virus removal can be monitored by time consuming infectivity assays or by fast quantitative assays such as real-time PCR or quantitative polymerase chain reaction (Q-PCR).
Q-PCR requires the proper negative and positive controls to be reliable. Nucleic acid extraction controls are added to test samples to control for proper nucleic acid extraction. If the nucleic acid extraction control is negative or outside of the expected recovery range during Q-PCR, then the sample is rejected. Conversely, if the nucleic acid extraction control is positive or within the expected recovery range during Q-PCR, nucleic acid extraction from the test sample is deemed reliable. A negative control is usually included in the Q-PCR assay, such as a sample-free buffer. The presence of a positive signal in the negative control might signify the contamination of the Q-PCR or nucleic acid extraction reagents with virus material.
A positive amplification control may also be included in a Q-PCR viral load assay. Such a positive control may include conserved viral nucleic acid sequences that are amplified using primers that amplify genuine viral contaminants. The failure of the Q-PCR to detect the positive control may indicate that the amplification procedure would have failed to detect a viral contaminant if one were present in the test sample.
The use of positive amplification controls, which emulate the target contaminant, creates its own problems. If the test sample is contaminated with even a slight amount of positive control, given the exquisite sensitivity of Q-PCR, the test sample may show a false positive. False positives can be mitigated to some extent by using a low level of positive amplification control, using segregated rooms for positive control work, using the UNG/dUTP system to selectively degrade PCR products containing dUTP, using single-use containers and displacement pipettes, and by thoroughly cleaning work areas and equipment. Regardless of the fastidious use of those mitigators, false positive results still occur during PCR testing.
In biopharmaceutical manufacturing, the risk of getting a false positive for a biological contaminant is non-negligible and may result in costly corrective actions. There is a great need for systems and methods to determine whether a given positive PCR result is a true positive or a false positive resulting from cross-contamination of the positive control. Applicants have developed and now disclose positive control compositions, systems and methods that permit the real-time determination of false positive Q-PCR signals.